Kit Size: 200 Assays (96-well) or 500 assays (384-well)
This kit is optimized to detect the activity of Hepatitis C Virus NS3/4A protease using an
EDANS/DABCYL FRET peptide substrate, with its fluorescence monitored at Ex/Em=340 nm/490nm upon proteolytic cleavage. It provides ample materials to perform 200 assays in a 96-well format and 500 assays in a 384-well plate.
Convenient Format: Complete kit including all the assay components.
Optimized Performance: Optimal conditions for the detection of HCV NS3/4A protease activity.
Enhanced Value: Less expensive than the sum of individual components.
High Speed: Minimal hands-on time.
The NS3/4A protease of Hepatitis C Virus (HCV) is required for the cleavage of viral
nonstructural polyprotein at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. These cleavages are essential for the maturation of the viral proteins. Thus, this protease has become one of the key targets for developing anti-HCV drugs.
The 490 HCV Protease Assay Kit provides a convenient assay for high throughput
screening of HCV NS3/4A protease inhibitors and for continuous quantification of HCV NS3/4A protease activity using fluorescence resonance energy transfer (FRET) peptide. The sequence of this FRET peptide is derived from the cleavage site of NS4A/NS4B. The cysteine on the natural cleavage site is replaced with aminobutyric acid (Abu) and the scissile amide bond with an ester bond. These modifications improved kcat/Km values by more than 100 fold and enabled the detection of the activity of NS3/4A protease at subnanomolar concentrations. In the FRET peptide, the fluorescence of EDANS is quenched by DABCYL. Upon cleavage into two separate fragments by HCV NS3/4A protease at the Abu-Ala bond (see Scheme 1), the fluorescence of EDANS is recovered, and can be monitored at excitation/emission = 340 nm/490 nm.
Components C and D can be stored at room temperature for convenience.
Component A: Microtiter Plate 1x200 wells HCV NS3/4A protease substrate (250ul)
EDANS/DABCYL FRET peptide, Ex/Em=340nm/490 nm upon cleavage
Component B: EDANS, fluorescence reference standard (100uM DMSO solution, 10ul)
Component C: 2X Assay buffer (25ml)
Component D: Stop solution (15ml)
Component E: DTT (1 M, 1ml)
Component F: Pep4AK (150uL, 600uM), HCV NS3 protease cofactor
Materials Required (but not provided):
96-well or 384-well microplate: Black microplate can provide better signal to noise ratio.
Fluorescence microplate reader: Capable of detecting emission at 490